1. Field of the Invention
This invention relates generally to taxonomic and phylogenic identification of organisms and specifically to the use of universal oligonucleotide primers and HSP60 amplicons to identify and distinguish organisms at the species level.
2. Description of Related Art
Coagulase positive and negative Staphylococci such as S. aureus and S. epidermidis are clinically important human pathogens (Jarvis and Martinec, J. Antimicrob. Chemother., 29(A):19-24, 1992; Kloos, and Lambe, Jr., Amer. Society of Microbiology, pp. 222-237, 1991). For example, coagulase negative Staphylococci are responsible for about 25% of nosocomial bacteremia in the United States (Banerjee, et al., Am. J. Med., 3B:86-89 (Suppl.), 1991). Like other bacteria, these Staphylococci species respond to external stress agents by expressing a set of heat shock proteins (HSP) (Ellis and van der Vies, Annu. Rev. Biochem., 60:321-347, 1991; Yura, et al., Annu. Rev. Microbiol., 47:321-360, 1993; Qoronfleh, et al., Antonie van Leeuwenhoek, 58:79-86, 1990). Of the known molecular weight classes of HSPs, the 60 kD and 70 kD families have been the most studied with the GroEL gene product from E. coli (Hemmingsen, et al., Nature, 333:330-334, 1988) being representative of the 60 kD family. This protein functions as a molecular chaperone with Gro ES (HSP 10) by assisting in protein folding (Gething and Sambrook, Nature, 355:33-45, 1992; Craig, et al., Cell, 78:365-372, 1994; Martin, et al., Nature, 366:228-233, 1993).
Previously studied methods for Staphylococcal identification using PCR amplification of either the 16S rDNA genes (Greisen, et al., J. Clin. Microbiol., 32:335-351, 1994) or spacer regions between 16S and 23S genetic loci as DNA targets (Jensen, et al., Appl. Environ. Microbiol., 59:945-952, 1994) failed to differentiate unequivocally some of the Staphylococcal species tested. The most promising nucleic acid based method for identification of the Staphylococci has been developed mainly by El Solh and colleagues (De Buyser, et al., J. Gen. Microbiol., 135:989-991, 1989; El Solh, et al., VCH Publishers, Inc., N.Y., pp. 585-593, 1990; De Buyser, et al., J. Gen. Microbiol., 138:889-899, 1992) and others (Thomson-Carter, et al., J. Gen. Microbiol., 135:2093-2097, 1989; Bialkowska-Hobrzanska, et al., Eur. J. Clin. Microbiol. Infect. Dis., 9:588-594, 1990; Pennington, et al., J. Clin. Microbiol., 29:390-392, 1991). The method is based on Southern blot analyses of 16S rRNA gene polymorphisms following restriction enzyme digestion of genomic DNA and gel separation of the digested DNA. However, this method is deemed laborious and requires pure bacterial cultures.
Other non-nucleic acid based commercial identification kits for laboratory identification of Staphylococci are also currently available (e.g., API Staph-Trac, API 20GP and Vitek GPI). However, recent publications report shortcomings in these test kits, especially when used for the identification of coagulase negative staphylococci (Grant, et al., Diagn. Microbiol. Infect. Dis., 18:1-5, 1994; Perl, et al., Diagn. Microbiol. Infect. Dis., 18:151-155, 1994). Several commercial products are available for the identification of isolates from the central nervous system (CNS), to the species level, however, very few of these systems have been evaluated and challenged in a direct comparative fashion using appropriate reference methods.
Three studies have reported the use of various Mycobacterium genus specific primers to amplify various target regions of the HSP 65 genes of Mycobacteria via PCR (Hance, et al., Mol. Microbiol., No. 3, 7:843-849, 1989; Plikaytis, et al., J. Clin. Microbiol., 30:1815-1822, 1992; Telenti, et al., J. Clin. Microbiol., 31:175-178, 1993). Hance, et al., used apparent species-specific complementary oligonucleotides to probe the PCR products. However, speciation was unsuccessful by this method.
Two other studies (Plikaytis, et al., supra; Telenti, et al., supra) described similar PCR strategies for Mycobacterial speciation, but these methods required the detection of restriction enzyme site polymorphisms (RFLP) within the PCR amplified products. Both methods relied on the use of two restriction enzymes for differentiating the Mycobacterial species. Also, intraspecies DNA RFLPs were observed. En initial studies with clinical samples (Telenti, et al., supra), problems were observed associated with generating sufficient PCR signals for visualizing DNA digestion products run on gels and with the presence of restriction enzyme inhibitors.
Consequently, there is an apparent need for a reliable and reproducible method for identifying and distinguishing Staphylococci species as well as other species of other genera. The present invention provides such a method.